Abstract
The hypothetical Escherichia coli protein YfhD has been identified as the archetype for the family 1B lytic transglycosylases despite a complete lack of experimental characterization. The yfhD gene was amplified from the genomic DNA of E. coli W3110 and cloned to encode a fusion protein with a C-terminal His(6) sequence. The enzyme was found to be localized to the outer membrane of E. coli, as would be expected for a lytic transglycosylase. Its gene was engineered for the production of a truncated soluble enzyme derivative lacking an N-terminal signal sequence and membrane anchor. The soluble YfhD derivative was purified to apparent homogeneity, and three separate in vitro assays involving high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were used to demonstrate the YfhD-catalyzed release of 1,6-anhydromuro-peptides from insoluble peptidoglycan. In addition, an in vivo bioassay developed using the bacteriophage lambda lysis system confirmed that the enzyme functions as an autolysin. Based on these data, the enzyme was renamed membrane-bound lytic transglycosylase F. The modular structure of MltF was investigated through genetic engineering for the separate production of identified N-terminal and C-terminal domains. The ability to bind peptidoglycan and lytic activity were only associated with the isolated C-terminal domain. The enzymatic properties of this lytic transglycosylase domain were found to be very similar to those of the wild-type enzyme. The one notable exception was that the N-terminal domain appears to modulate the lytic behavior of the C-terminal domain to permit continued lysis of insoluble peptidoglycan, a unique feature of MltF compared with other characterized lytic transglycosylases.
Highlights
Lytic transglycosylases (LTs) are a class of autolysins that play an integral role in the metabolism of the PG sacculus
The hypothetical E. coli protein YfhD has been identified as the archetype for the family 1B LTs based on sequence alignments and the presence of consensus motifs [4]
We present data that confirms YfhD does function as an LT, and based on this evidence, we propose to rename it membrane-bound lytic transglycosylase F (MltF)
Summary
DNase I, RNase A, Pronase, IPTG, and EDTA-free protease inhibitor tablets were purchased from Roche Applied Science. Examination and comparison of various E. coli genome sequences revealed the possibility of a start codon further upstream encoding a protein with an N-terminal signal sequence This extended yfhD ORF and its associated truncated form (lacking the N-terminal signal sequence) were amplified with the same downstream primer EsR28 but using different upstream primers, EsF28full and EsF28trunc, respectively. Amplified ORFs were cleaned using a PCR clean up kit (Roche Applied Science), digested with HindIII and NcoI, ligated with appropriately digested pET28a(ϩ) plasmid DNA, and transformed into E. coli DH5␣. Following plasmid amplification by PCR, products were digested overnight with DpnI and transformed into E. coli DH5␣, and selected colonies were screened for loss of the NcoI site through analytical restriction enzyme digests and nucleotide sequencing. In place of residues in place of residues Lys-182 to Lys-421;GmR, CmR aacC1gene (GmR) from pUCGm ligated into the EcoRV site of pPS584; GmR cassette is flanked by identical inverted MCS pET30a(ϩ) derivative containing mltB on an NdeI/XhoI fragment encoding an N-terminal truncation of Arg-2 to Cys-17; KmR
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